Depigmentation is the lightening of the skin, or loss of pigment. Depigmentation of the skin can be caused by a number of local and systemic conditions. The pigment loss can be partial, for instance injury to the skin, or complete, such as in vitiligo, temporary or permanent.
Depigmentation of the skin is a disease landmark in people affected by vitiligo, which produces differing areas of light and dark skin. Next to the cosmetic disadvantages, skin areas with low pigmentation are prone to sun burns or even more adverse effects, which, in the worst case, result in skin cancer. Hence, substantial efforts are currently undertaken to provide an easy and reliable method for protecting these depigmented areas by providing pigmentation. One approach is to apply melanocytes on the depigmented skin areas. Melanocytes are melanin-producing cells, inter alia, located in the bottom layer (the stratum basale) of the skin epidermis. Melanin is the pigment that primarily determines the color of the skin and enables its primary protection from sun radiation.
Existing medical treatments for vitiligo are partially helpful and are either palliative or invasive. Therefore, a high health interest and market potential exist for a causative, autologous, non-invasive treatment.
The developmental potential of the hair follicle outer root sheath (ORS), which contains pluripotent adult stem cells, transiently amplifying cells, precursors and differentiated cells, is known. These cells are capable of giving rise to melanocytes (among other cell types). Several methodological upgrades have been addressing duration, yield and culture purity since the first long-term cultivation of human hair follicle melanocytes (HM) in 1995 (Tobin D J, Colen S R, Bystryn J C. J Invest Dermatol 1995: 104: 86-89).
However, there is a need for methods to isolate and differentiate melanocyte-yielding cells in order to retrieve pure melanocytes for further processing and application on the skin. Furthermore, there is a need to provide a method to increase the number of yielded melanocytes after a short period of culture. The inventors now provide means and methods as disclosed herein below solving that problem. The inventors furthermore, found that such melanocytes are very suitable for treatment of depigmentation and are able to show unexpectedly high melanin production and a high enzymatic efficacy in three-dimensional structures.
There are methods known how to obtain and cultivate melanocytes. One approach is the differentiation and cultivation of melanocytes from stem cells from hair follicles (WO-A2 2009/049734). For such purpose, epilated hairs are treated enzymatically to release the stem cells from the hair follicle. After differentiation and cultivation, application to the affected areas was suggested. However, the inventors found that by the method disclosed in WO-A2 2009/049734 also cell types that are surplus to requirements of the treatment, such as fibroblasts and keratinocytes, are cultivated which show adverse effects on the efficacy of the treatment. With known methods the superfluous cell types are also being cultivated, which leads to a loss of efficacy of the depigmentation therapy or makes it more complicated at its best. Hence, there is a need for improved methods to generate and obtain melanocytes. Moreover, there is a need to provide such cells with high purity.
Dieckmann et al. (2010); Experimental Dermatology; 19(6):543-545 discloses a non-invasive method for obtaining these adult stem cells from outer root sheath (ORS) of a plucked hair follicle. However, Dieckmann (loc. cit.) does not disclose (1) that the bulb of an epilated human hair is removed and the remaining part of the epilated hair be used; and (2) that the epilated hair are treated with collagenase. In addition, the cultivation in accordance with the herein provided method can comprise a step of selecting and/or isolating melanocytes comprising Geneticin treatment. Such a step is not disclosed in Dieckmann (loc. cit.).
As shown in Example 2, the method of the present invention allows for a much more pronounced growth and proliferation of melanocytes as compared to the method of Dieckmann (loc. cit.). FIG. 15 shows that the herein provided method provides an exponential increase in the cell number over several passages, whereas the cell number even decreases if the Dieckmann (loc. cit.) method is used. As demonstrated in Example 2, the Dieckmann (loc. cit.) method did not allow for the production of more than 710,000 melanocytes in total whereas the method of the present invention allowed the production of about 80,875,000 melanocytes in total after 6 passages under otherwise comparable conditions. In other words, the present invention provides for a more than 100-fold higher production of melanocytes as compared to the Dieckmann method (loc. cit.). If the corresponding numbers of generated melanocytes per epilated human hair (or likewise per follicle) are calculated, the Dieckmann (loc. cit.) method did not allow for the production of more than about 7,000 (more exactly 7,717 melanocytes) per epilated human hair (or per follicle), whereas the method of the present invention allowed the production of about 2,700,000 melanocytes per epilated human hair (or per follicle) after 6 passages under otherwise comparable conditions. In other words, the present invention provides for a more than 100-fold higher production of melanocytes as compared to the Dieckmann method (loc. cit.). The method of the present invention can advantageously comprise adherent culture of melanocytes.
A first difference of the herein provided method compared to the Dieckmann (loc. cit.) method is the removal of the bulb of an epilated human hair. It is believed that the bulb contains and carries over major amounts of fibroblasts. By removing the bulb, the epilated hair to be used herein contains/yields less fibroblasts. Thus, there are less fibroblasts at the very start of the herein provided method as compared to Dieckmann (loc. cit.). It is believed that this allows a better growth of melanocytes, because the cell culture is less contaminated with fibroblasts and there is therefore less competition for nutrients and space from the very beginning on.
A second difference of the herein provided method as compared to Dieckmann (loc. cit.) is the incubation of the epilated hair with a collagen degrading agent (like Collagenase). Said incubation can take 10 minutes. The collagen degrading agent loosens the extracellular matrix of the epilated hair. It is believed that said loosening facilitates the leave or migration of stem cells from the extracellular matrix. Thus, the incubation with a collagen degrading agent can separate stem cells from the outer root sheath. As demonstrated in Example 2, FIGS. 13 and 14, the Outer Root Sheath surface/cell number is indeed increased as compared to Dieckmann (loc. cit.), if the method of the present invention is employed. Indeed, the Outer Root Sheath surface/cell number was almost twice as high (factor 1.84) as compared to Dieckmann (loc. cit.).
It is believed that due to that the reduction of fibroblasts and easier/quicker/increased leave of stem cells from the extracellular matrix have the advantageous and surprising effect that the present method allows for a pronounced increase in the growth and development of melanocytes in accordance with the present invention, as demonstrated in Example 2. Dieckmann (loc. cit.) provided no hint to the removal of the bulb or collagenase treatment, let alone any advantages conferred thereby. To the contrary, Dieckmann (loc. cit.) even taught away from enzymatic treatment, because it was potentially unfavourable for the cells.
As explained below, the Geneticin treatment is a further advantageous aspect of the herein provided method. As shown in FIG. 15/16 the use of Geniticin in the method of Dieckmann (loc. cit.) does, in contrast to the herein provided method, not allow for a substantial growth or selection/isolation of melanocytes. The Geneticin treatment in accordance with the present method is advantageous because it targets primarily cells with a rapid metabolism and proliferation, such as fibroblasts and keratinocytes, whereas it hardly affects slowly-dividing melanocytes. Thus, the Geneticin treatment allows for an enrichment of melanocytes in the cell culture and a pronounced increase in melanocyte number. Geneticin treatment in the Dieckmann (loc. cit.) method does not show any such desirable effects. Therefore, it is believed that the cell culture according to Dieckmann (loc. cit.) prior to Geneticin treatment contains, in contrast to the method of the present invention, substantial amounts of fibroblasts and keratinocytes and only a minor amount of melanocytes. Thus, it is believed that the amount of melanocytes after Geneticin treatment of the Dieckmann (loc. cit.) cell culture is not sufficient for efficient selection/isolation and growth of melanocytes.
As explained above, the inventors now developed an improved non-invasive method for obtaining adult stem cells from outer root sheath (ORS) of a plucked hair follicle and differentiation into a pure culture of functional melanocytes. The melanocytes prepared by the method according to the present invention as set out herein can be readily used for treatment of Vitiligo as suspension, liquid or in the form of an aerosol, as single culture or combined with keratinocytes. The melanocytes are further provided in autografts, homografts or allografts together with keratinocytes. The grafts provided are stabilized by biocompatible (scaffold) carriers.
The present invention now allows to obtain melanocytes non-invasively, through differentiation from stem cells from the outer root sheath of epilated human hairs in high quantity and purity, in a small time frame of 4 weeks and less.